Abstract
NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.
Highlights
MATERIALS AND METHODSReagents and Antibodies—The pcDNA4/HisMaxC plasmid used for the construction of the expression vector was from Invitrogen (Carlsbad, CA)
Despite the critical role of NEK6 in maintaining proper progression of the cell cycle, the physiological substrates of NEK6 are largely undefined
We demonstrate that NEK6 is overexpressed in various human cancer tissues, and ectopic expression of NEK6 increases tumor promoter-induced transformation of JB6 Cl41 mouse epidermal cells
Summary
Reagents and Antibodies—The pcDNA4/HisMaxC plasmid used for the construction of the expression vector was from Invitrogen (Carlsbad, CA). NEK6 Induces Cell Transformation constructed expression vectors were confirmed by restriction mapping and DNA sequencing. C, differential expression of NEK6 in normal and cancer cell lines. Proteins were isolated from different cell lines, and the expression of NEK6 was analyzed by Western blot. NEK6 was constructed and optimized with the program Prime version 2.1 (Schrodinger, LLC, New. York) based on the sequence alignment with the known crystal structure of active MAP3K TAO2 kinase (Protein Data Bank code 2GCD). A, stable transfectants of JB6 Cl41 cells expressing mock (pcDNA4-mock), wild type (pcDNA4-NEK6), or mutant (pcDNA4-NEK6 K74M/K75M) NEK6 were established, and overexpression was confirmed by Western blot using an antibody to detect the Xpress tag. C and D, NEK6 expression increases EGF-induced (C) or TPA-induced (D) anchorage-independent cell transformation. The cultures were maintained in a 37 °C, 5% CO2 incubator for 10 days (EGF) or 3– 4 weeks (TPA), and the cell colonies that 86.4% of all residues were in favored (98%) regions and that 97.9% were in allowed regions (Ͼ99.8%), suggesting a reliable molecular arrangement
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